The earliest method of protein display is phage display (Smith, 1985), in which the protein of interest is fused to one of the outer-coat proteins of the phage where it may be present along with wild-type copies of the protein. For example, a display platform based on the M13 filamentous phage using fusions to the pIII protein.
Other display methods include ‘in vitro’ display methods where the protein is expressed using a cellular translation extract, and the coupling between the protein and the coding nucleic acid is achieved through physical linkage (e.g. ribosome display, mRNA display) or through attachment to a common scaffold or encapsulation within a membrane, such as in in vitro compartmentalization (IVC) where the mRNA is translated within a micelle suspension that may also include a microbead (magnetic or sepharose) capture system for both mRNA and protein.
Another method of protein display is microbial surface display which involves the targeted location of expressed proteins to the exterior of a microbial cell, either gram-negative, gram-positive eubacteria or yeast. The proteins are fused to anchor domains that attach them to the cell surface. The anchor domains may have motifs dictating lipidation or covalent attachment to the cell wall, or they may be a fusion to an integral membrane protein within an exposed loop region. Due to the scalability of production, microbial surface display may not only be used for screening for improved protein variants from a diverse library, but may also be used to present antigens for vaccination or as a cellular-scaffold for enzymes for industrial biotechnology.
Protein display methods are commonly applied to the evolution of affinity proteins, such as antibodies. Single molecule display methods are historically the most popular, but they suffer from high background and low resolution between affinity scales. Proteins identified by surface display in yeast or by phage systems are usually reformatted for expression in the E. coli periplasm, even though periplasmic yields are often extremely poor comparable to expression in the cytoplasm. When antibodies are expressed in the cytoplasm at high yield, however, in almost every instance they form insoluble inclusion bodies that must be laboriously refolded and tested for activity.
Thus, there remains a need for methods of protein display, particularly for the screening of affinity protein display libraries and enzyme libraries.